Cyclic hydroxylamine as psychoactive compounds

ABSTRACT

A compound of formula (I): in which X represents O or CH 2 ; R 3  and R 4  each independently represent hydrogen or C 1-6  alkyl; p represent 0 or 1; and R represents a five- or six-membered saturated or unsaturated ring selected from: formula (II), (III) and (IV); or R represents a five- or six-membered oxo-substituted unsaturated ring selected from: (V) and (VI); wherein R 1  and R 2  together represent an oxo group, or R 1  and R 2  each represent hydrogen, methoxy or ethoxy, or R 1  and R 2  together with the interjacent carbon atom represent a 1,3-dioxolane or 1,3-dioxane ring, attached via the 2 position and optionally bearing one or more methyl or ethyl groups; or a salt thereof, is suitable for the treatment of anxiety and depression.

This application is a continuation of application Ser. No. 10/559,180filed Feb. 2, 2006, now U.S. Pat. No. 7,842,689, which is a 371 ofPCT/GB2004/002324 filed Jun. 1, 2004, and claims priority to BritishApplication Nos. 0313628.0 filed Jun. 12, 2003, and 0328439.5 filed Dec.8, 2003, the entire contents of each of which are hereby incorporated byreference.

This invention relates to psychoactive compounds suitable for thetreatment of anxiety and depression. In particular, this inventionrelates to novel hydroxylamine compounds such as ring-opened derivativesof isoxazole compounds and their analogues, their preparation,pharmaceutical formulations thereof and their use in medicine.

A number of psychoactive compounds are known for use in the treatment ofanxiety and depression. Diazepam (a benzodiazepine) is well-known, andwidely used as an anxiolytic and anti-depressant. Other knownpsychoactive compounds include certain tricyclic fused benzo[d]isoxazolecompounds having the structure shown below in formula (A), as disclosedin U.S. Pat. No. 5,707,988 (Boyd et al./British Technology Group Ltd.):

in which X′ is O, S, C═O or NR′ wherein R′ is hydrogen, C₁₋₆ alkyl,phenyl or C₇₋₁₂ phenalkyl, R₁′ and R₂′ each represent hydrogen ortogether represent an oxo group and R₃′, R₄′ and R₅′ each representhydrogen or R₁′ represents hydrogen and two of R₂′, R₃′, R₄′ and R₅′together represent the second bond of a double bond joining positions 7and 8, 8 and 9 or 9 and 10 with the remaining two of R₂′, R₃′, R₄′ andR₅′ representing hydrogen, or a salt thereof.

It has been found that, although the compounds of formula (A)demonstrate good efficacy as anxiolytic agents, their efficacy in thetreatment of depression is less than ideal. There is therefore a needfor new compounds that are effective in the treatment of both anxietyand depression, in particular for the treatment of pathologies in whichboth conditions occur. The present invention addresses that need.

The following compounds are known as synthetic intermediates:

2-Phenylpiperidin-1-ol is mentioned in the following papers:

-   “Regiochemistry of mercury(II) oxide oxidation of unsymmetrical    N,N-disubstituted hydroxylamines.” Tetrahedron (1996), 52(47),    14917-28 (Ali et al.);-   “Reaction of pyridine and quinoline N-oxides with phenylmagnesium    bromide.” Y. Org. Chem. (1965), 30(3), 910-13 (Kato et al.);-   “Cyclic nitrones (I): dimeric 2,3,4,5-tetrahydropyridine N-oxides.”    Chem. Ber., vol. 89, 2159-67 (1956) (Thesing et al.).

2-Benzylpiperidin-1-ol is mentioned in the following papers:

-   “Synthesis and cycloaddition of 6-substituted    3,4,5,6-tetrahydropyridine 1-oxides.” J. Chem. Res., Synop. (1994),    (2), 54-5 (Ali et al.);-   “Mercury(II) oxide oxidation of 2-substituted N-hydroxypiperidine: a    solution to the regiochemical problem.” Tet. Lett. (1993), 34(33),    5325-6 (Ali et al.).

However, there is no disclosure of the compounds for use aspharmaceuticals.

Accordingly, the present invention provides in a first aspect a compoundof formula (I):

in which—

X represents O or CH₂;

R³ and R⁴ each independently represent hydrogen or C₁₋₆ alkyl;

p represent 0 or 1; and

R represents a five- or six-membered saturated or unsaturated ringselected from:

or

R represents a five- or six-membered oxo-substituted unsaturated ringselected from:

wherein R¹ and R² together represent an oxo group, or R¹ and R² eachrepresent hydrogen, methoxy or ethoxy, or R¹ and R² together with theinterjacent carbon atom represent a 1,3-dioxolane or 1,3-dioxane ring,attached via the 2 position and optionally bearing one or more methyl orethyl groups;

or a tautomer thereof;

or a salt thereof;

for use as a pharmaceutical.

In the case where R represents a five- or six-membered oxo-substitutedunsaturated ring selected from:

wherein R¹ and R² together represent an oxo group, or an acetal thereof,wherein R¹ and R² each represent methoxy or ethoxy, or R¹ and R²together with the interjacent carbon atom represent a 1,3-dioxolane or1,3-dioxane ring, attached via the 2 position and optionally bearing oneor more methyl or ethyl groups, then preferably p represents 0. Theinvention thus preferably embraces compounds of formula (IA), (IB), (IC)(ID), (IE) and (IF):

as well as ketones of formula (IG) and (IH):

and acetals of the ketones of formula (IG) or (IH) with methanol orethanol; or with ethane-1,2-diol or propane-1,2-diol, optionally bearingone or more methyl or ethyl groups.

When R¹ and R² together with the interjacent carbon atom represent a1,3-dioxolane or 1,3-dioxane ring bearing one or more methyl or ethylgroups, preferably one methyl or ethyl group is positioned adjacent toeach of the oxygen atoms in the 1,3-dioxolane or 1,3-dioxane ring.Preferably these are in the trans orientation, giving a compound suchas:

Such compounds might be hydrolysed in vivo to produce the correspondingcompound of formula (I) in which R¹ and R² together represent an oxogroup. The compounds having the 3-dioxolane or 1,3-dioxane ringtherefore represent potential prodrugs.

However, preferably R¹ and R² together represent an oxo group.

In the case where R represents a five- or six-membered oxo-substitutedunsaturated ring selected from:

then preferably X is O.

Synthesis is facilitated if the heterocyclic ring demonstrates somesymmetry Thus preferably R³ and R⁴ are identical. Preferably R³ and R⁴each represents hydrogen.

In a second aspect, the present invention provides a compound of formula(II):

in which—

R³ and R⁴ each independently represent hydrogen or C₁₋₆ alkyl;

p represent 0 or 1; and

R represents a five- or six-membered saturated or unsaturated ringselected from:

or

R represents a five- or six-membered oxo-substituted unsaturated ringselected from:

wherein R¹ and R² together represent an oxo group, or R¹ and R² eachrepresent hydrogen, methoxy or ethoxy, or R¹ and R² together with theinterjacent carbon atom represent a 1,3-dioxolane or 1,3-dioxane ring,attached via the 2 position and optionally bearing one or more methyl orethyl groups;

or a tautomer thereof;

or a salt thereof.

A further preferred embodiment of the present invention is a compound offormula (III):

in which—

R¹ and R² each represent hydrogen or together represent an oxo group;

or a salt thereof.

In this specification the term “alkyl” includes both straight andbranched chain groups, as well as saturated and unsaturated groups.

When R¹ and R² together represent an oxo group, certain compounds of theinvention might exist as tautomers where the N-hydroxyl group hasreacted with the carbonyl group to form a hemiacetal of formula (IV) or(V):

Thus the hemiacetals of formula (IV) and (V) are within the scope of theinvention.

As indicated above, the compounds of the invention may exist in the formof a salt, preferably an amine salt. Such salts may be formed with aphysiologically acceptable inorganic or organic acid. Physiologicallyacceptable acids include hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, isethionic, acetic, fumaric, maleic, salicylic,p-toluenesulfonic, tartaric, citric, lactobionic, formic, malonic,pantothenic, succinic, naphthalene-2-sulfonic, benzenesulfonic,methanesulfonic and ethanesulfonic acid. Hydrochloric acid is preferred.However, in general it is preferred to use the free base rather than thesalt.

The compounds of the invention possess a chiral centre (denoted *) atthe carbon atom adjacent the nitrogen of the N—OH group. It will beappreciated that the compounds of the invention can be resolved intotheir enantiomeric forms, or exist as a racemate. Depending on thenature of the substituents, a number of diastereoisomers is alsopossible.

In the case where p represents 0 and R represents a five- orsix-membered oxo-substituted unsaturated ring selected from:

the compounds of the invention may be prepared by the reaction of acompound of formula (VI) or (VIA) with a compound of formula (VII):

in which R¹, R² and X are as defined with respect to formula (I). Itwill be appreciated that this will be most straightforward when R¹ andR² together represent an oxo group, as the double bond compound offormula (VI) or (VIA) will then be activated by the presence of thecarbonyl group. Other electron-withdrawing groups would also facilitatereaction.

The reaction is preferably carried out by combining the compounds offormulae (VI) or (VIA) and (VII) at a temperature in the range of fromroom temperature to 100° C., and then heating the reaction mixture to atemperature in the range of from 50 to 150° C., more preferably 55 to65° C. for several hours in a suitable solvent. Ambient pressure may beused, but preferably the reaction is carried out at ultrahigh pressurein a sealed tube. The compound of formula (VI) or (VIA) may itself be asuitable solvent for the reaction; for example, in the case where R¹ andR² together represent an oxo group, the resultant 2-cyclohexenone and2-cyclopentenone are both readily available.

The compound of formula (I) is thereby produced via the formation of atricyclic intermediate of formula (VIII) or (VIIIA):

which may be isolated or left to remain in situ. Protonation of theintermediate (VIII) or (VIIIA), or a corresponding base-catalysedreaction, leads to the formation of compound (I). Ring openingpresumably appears by an E1cb elimination (“cb” denoting “conjugatebase”), or the corresponding acid-catalysed reaction, preferably atelevated temperature.

The compound of formula (VII) is preferably formed in situ from acompound of formula (IX):

via oxidation, preferably a pertungstate-catalysed oxidation. Thenitrone (VII) has a tendency to polymerise and, if it needs to beisolated, care should be taken to avoid polymerise happening by storingin a freezer.

Any of the compounds of the invention may also be prepared by thereaction of a Grignard reagent of formula R(CH₂)_(p)MgHal with acompound of formula (VII):

in which Hal represents halide and R, R¹, R² and X are as defined withrespect to formula (I). We have fund that the Grignard reagents preparedusing a chloride anion to be perfectly satisfactory.

The reaction is preferably carried out by combining the Grignard reagentof formula R(CH₂)_(p)MgHal with the compound of formula (VII) at atemperature of −10° C., and then holding the reaction mixture at atemperature of 0° C. in a suitable solvent. Tetrahydrofuran is asuitable solvent for the reaction.

It will be appreciated that the compounds of formula (I) may also beprepared by modifications of these processes and by other alternativeprocesses, which will be apparent to a person skilled in the chemicalart.

The compounds of the invention have been found to effective in thetreatment of both anxiety and depression. They are particularly usefulfor the treatment of anxiogenesis caused by withdrawal frombenzodiazepines (as they exhibit cross tolerance with thesebenzodiazepines in comparison with buspirone, for example, which doesnot). The compounds are also of use in the treatment of anxiogenesiscaused by abruptly ceasing the administration of drugs of abuse such asnicotine, alcohol and cocaine.

The dosage level of the compounds of formula (I) required to achieveeffective anxiolysis or anti-depressant activity will vary with themammal treated and will depend on factors such as the mammal's bodyweight, its surface area, age and general state of health. It will alsodepend upon the mode of administration. Dosage levels of 0.01 mg/kg to100 mg/kg, particularly 1 mg/kg to 10 mg/kg, are suitable. Dependingupon the nature and severity of the condition being treated, the dosesmay be repeated up to 2 or 3 times per day during the period oftreatment. Doses outside these ranges may be administered ifappropriate.

The compounds of formula (I) may be administered using oral, rectal,parenteral, subcutaneous or topical routes.

The compounds of formula (I) may be administered alone or together witha pharmaceutically acceptable carrier, such as an excipient or diluent.The invention therefore further provides a pharmaceutical compositioncomprising a compound of formula (I) in association with apharmaceutically acceptable carrier therefor. The composition mayfurther comprise additional therapeutic agent(s) or ingredient(s).

The compounds and compositions of the invention may conveniently bepresented as unit dosage forms prepared using techniques that are wellknown to a person skilled in the art. In general, preparation of theunit dosage form includes the step of bringing one or moretherapeutically active compounds into association with the carrier. Theactive compound(s) or and/or carrier ingredient(s) are preferably in theform of a finely divided solid or a liquid.

Compositions suitable for oral administration include discreet unitssuch as tablets, capsules, caplets, cachets or lozenges, each containinga predetermined amount of the therapeutic compound. Solutions andsuspensions of the therapeutic compound in an aqueous or a non-aqueousliquid are also suitable for oral administration and include syrups,elixirs and emulsions. The compound may also be presented as a bolus,electuary or paste.

Administration of the compound by a parenteral routes includesintravenous, intraperitoneal, intra-muscular and intra-articularadministration. Compositions suitable for parenteral administrationconveniently include a sterile aqueous preparation of the activecompound, suitable for injection or infusion

Compositions suitable for topical administration include lotions, creamsand pastes. The compositions of the invention may also be presented inthe form of an aerosol or a suppository.

Accordingly the present invention also provides a method for preventingor alleviating the symptoms of anxiety and/or depression, which methodcomprises administering to a patient in need of such treatment,particularly a warm-blooded animal such as a human, a non-toxic,therapeutically effective amount of a compound of a compound of formula(I), or a salt thereof, or a composition containing such a compound.

The invention also includes a compound of formula (I) for use as amedicament, and the use of a compound of formula (I), or a salt thereof,in the manufacture of a medicament for treatment of both anxiety anddepression, particularly for the treatment of anxiogenesis caused bywithdrawal from benzodiazepines, or caused by abruptly ceasing theadministration of drugs of abuse such as nicotine, alcohol and cocaine.

The invention will now be described by reference to the followingExamples. Variations on these Examples falling within the scope of theinvention will be apparent to a person skilled in the art.

EXAMPLES Example 1 Preparation of2-(4-hydroxy-3-morpholinyl)-2-cyclohexenone

Morpholine (10.97 g; 0.126 M) and sodium tungstate hydrate (1.54 g; 4.67mM) were cooled to 0° C. in a 250 mL flask. Hydrogen peroxide (32.5 mL(30% aq.); 0.286 mM) was slowly added, keeping the reaction temperatureat 0° C. The reaction mixture was stirred for a further 1.5 h and excesshydrogen peroxide was destroyed using sodium bisulfate. 2-Cyclohexenone(12.1 mL; 0.12 5M) was added slowly to the flask, and the reactionmixture was stirred for a further 48 h. The reaction mixture was heatedto 55° C. for 2 h and then 65° C. for a further 2 h. The reactionmixture was then poured into aqueous sodium chloride and was extractedwith dichloromethane (3×100 mL). The organic layer was washed withsaturated sodium bicarbonate solution, separated and dried overmagnesium sulfate. Removal of the solvent gave a light brown oil, whichwas subjected to Kugelrohr distillation at 50-60° C. under vacuum,producing a dark oil (3.5 g; 14%). The compound was further purified bychromatography on a silica column using a dichloromethane(CH₂Cl₂):acetonitrile (CH₃CN) eluent (51; 0.27).

ν_(max)(cm⁻¹)=3435 (OH); 2958, 2941, 2916, 2901, 2874 (CH); 2777w; 1699s(C═O); 1476; 1101.

Acc. Mass spectrometry: calculated=198.1130 (MH⁺); found=198.1130.

Example 2 Preparation of 3-phenylmorpholin-4-ol

Activated MnO₂ (0.65 g, 7.41 mmol) was added to a solution ofN-hydroxymorpholine (0.25 g, 2.47 mmol) in dichloromethane (15 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of phenylmagnesium chloride (2.0 M intetrahydrofuran, 2.47 mL, 4.94 mmol) at −10° C. The reaction mixture wasstirred at 0° C. for 0.5 h and then saturated aqueous ammonium chloridesolution (15 mL) and dichloromethane (15 mL) were added. The aqueousphase was separated and extracted with dichloromethane (2×20 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The crude product was subjected to flashchromatography on SiO₂ eluting with 25% diethyl ether in hexanes to givethe title compound (0.045 g, 11%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3201 (br., O—H), 1104 (s, C—O)

δ_(H) (300 MHz): 7.22 (m, 5H), 3.85 (d, 1H, J=10.5 Hz), 3.72-3.50 (m,3H), 3.31 (t, 1H, J=10.5 Hz), 3.12 (d, 1H, J=10.5 Hz), 2.81 (td, 1H,J₁=11.2 Hz, J₂=3.4 Hz).

δ_(C) (75.5 MHz): 138.1 (0), 128.6 (1), 127.9 (1), 72.45 (2), 72.16 (1),66.78 (2), 57.67 (2).

HRMS (electrospray mode): m/z=180.1026710 (MH⁺) (calc. 180.1019051).

Example 3 Preparation of 3-benzylmorpholin-4-ol [P2]

Activated MnO₂ (1.29 g, 14.8 mmol) was added to a solution ofN-hydroxymorpholine (0.50 g, 4.95 mmol) in dichloromethane (25 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of benzylmagnesium chloride (2.0 M intetrahydrofuran, 4.95 mL, 9.90 mmol) at −10° C. The reaction mixture wasstirred at 0° C. for 0.5 h and then saturated aqueous ammonium chloridesolution (25 mL) and dichloromethane (25 mL) were added. The aqueousphase was separated and extracted with dichloromethane (2×30 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The crude product was subjected to flashchromatography on SiO₂ eluting with 25% diethyl ether in hexanes to givethe title compound (0.326 g, 34%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3203 (br., O—H), 1107 (s, C—O).

δ_(H) (300 MHz): 7.23 (m, 5H), 3.89 (d, 1H, J=11.7 Hz), 3.74-3.56 (m;2H), 3.44 (d, 1H, J=13.0 Hz), 3.23 (m, 2H), 2.87 (m, 2H), 2.39 (t, 1H,J=10.5 Hz).

δ_(C) (75.5 MHz): 137.7 (0), 129.2 (1), 128.5 (1), 126.4 (1), 70.22 (2),67.76 (1), 66.60 (2), 58.60 (2), 35.85 (1).

HRMS (electrospray mode)=194.1186060 (MH⁺) (calc. 194.1175552).

Example 4 Preparation of 3-cyclohexylmorpholin-4-ol

Activated MnO₂ (1.29 g, 14.8 mmol) was added to a solution ofN-hydroxymorpholine (0.50 g, 4.95 mmol) in dichloromethane (25 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of cyclohexylmagnesium chloride (2.0 M in diethylether, 4.95 mL, 9.90 mmol) at −10° C. The reaction mixture was stirredat 0° C. for 0.5 h and then saturated aqueous ammonium chloride solution(25 mL) and dichloromethane (25 mL) were added. The aqueous phase wasseparated and extracted with dichloromethane (2×30 mL). The combinedorganic layers were dried (Na₂SO₄) and concentrated under reducedpressure. The crude product was subjected to flash chromatography onSiO₂ eluting with 50% diethyl ether in hexanes to give the titlecompound (0.220 g, 24%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3203 (br., O—H), 1107 (s, C—O).

δ_(H) (300 MHz): 3.84 (m, 1H), 3.55 (t, 1H, J=11.3 Hz), 3.17 (d, 1H,J=10.5 Hz), 2.79 (t, 1H, J=10.5 Hz), 2.46 (d, 1H, J=9.8 Hz), 2.06-1.45(m, 6H), 1.35-0.82 (m, 6H).

δ_(C) (75.5 MHz): 70.83 (1), 67.42 (2), 66.29 (2), 58.84 (2), 36.71 (1),30.14 (2), 27.64 (2), 26.96 (2), 26.75 (2).

HRMS (electrospray mode)=186.1496690 (MH⁺) (calc. 186.1488553).

Example 5 Preparation of 3-cyclopentylmorpholin-4-ol

Activated MnO₂ (1.29 g, 14.8 mmol) was added to a solution ofN-hydroxymorpholine (0.50 g, 4.95 mmol) in dichloromethane (25 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of cyclopentylmagnesium chloride (2.0 M indiethyl ether, 4.95 mL, 9.90 mmol) at −10° C. The reaction mixture wasstirred at 0° C. for 0.5 h and then saturated aqueous ammonium chloridesolution (25 mL) and dichloromethane (25 mL) were added. The aqueousphase was separated and extracted with dichloromethane (2×30 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The crude product was subjected to flashchromatography on SiO₂ eluting with 70% diethyl ether in hexanes to givethe title compound (0.246 g, 29%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3233 (br., O—H), 1118 (s, C—O).

δ_(H) (300 MHz): 3.90 (t, 2H, J=9.9 Hz), 3.55 (t, 1H, J=10.7 Hz), 3.30(t, 1H, J=10.3 Hz), 3.15 (d, 1H, J=11.1 Hz), 2.80 (t, 1H, J=10.4 Hz),2.57 (m, 1H), 2.19 (m, 1H), 1.89 (m, 1H), 1.73-1.02 (m, 7H)

δ_(C) (75.5 MHz): 69.31 (1), 67.91 (2), 65.65 (2), 57.83 (2), 39.91 (1),30.80 (2), 28.19 (2), 24.89 (2).

HRMS (electrospray mode)=172.1327720 (MH⁺) (calc. 172.1332052)

Example 6 Preparation of 2-phenylpiperidin-1-ol

Activated MnO₂ (1.29 g, 14.8 mmol) was added to a solution ofN-hydroxypiperidine (0.50 g, 4.95 mmol) in dichloromethane (25 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of phenylmagnesium chloride (2.0 M intetrahydrofuran, 4.95 mL, 9.90 mmol) at −10° C. The reaction mixture wasstirred at 0° C. for 0.5 h and then saturated aqueous ammonium chloridesolution (25 mL) and dichloromethane (25 mL) were added. The aqueousphase was separated and extracted with dichloromethane (2×30 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The crude product was subjected to flashchromatography on SiO₂ eluting with 30% diethyl ether in hexanes to givethe title compound (0.330 g, 38%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3204 (br., O—H).

δ_(H) (300 MHz): 7.30 (m, 5H), 3.34 (m, 2H), 2.59 (t, 1H, J==11.0 Hz),1.87-1.50 (m, 3H), 1.36 (m, 1H).

δ_(C) (75.5 MHz): 143.4 (0), 128.4 (1), 127.4 (1), 127.1 (1), 73.51 (1),58.94 (2), 35.21 (2), 25.83 (2), 24.07 (2).

HRMS (electrospray mode)=178.1223660 (MH⁺) (calc. 178.1226406).

Example 7 Preparation of 2-benzylpiperidin-1-ol

Activated MnO₂ (1.29 g, 14.8 mmol) was added to a solution ofN-hydroxypiperidine (0.50 g, 4.95 mmol) in dichloromethane (25 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of benzylmagnesium chloride (2.0 M intetrahydrofuran, 4.95 mL, 9.90 mmol) at −10° C. The reaction mixture wasstirred at 0° C. for 0.5 h and then saturated aqueous ammonium chloridesolution (25 mL) and dichloromethane (25 mL) were added. The aqueousphase was separated and extracted with dichloromethane (2×30 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The crude product was subjected to flashchromatography on SiO₂ eluting with 60% diethyl ether in hexanes to givethe title compound (0.309 g, 33%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3212 (br., O—H).

δ_(H) (300 MHz): 7.24 (m, 5H), 3.65 (d, 1H, J=10.5 Hz), 3.40 (d, 1H,J=10.4 Hz), 2.66-2.48 (m, 2H), 2.40 (t, 1H, J=11.2 Hz), 1.79-1.48 (m,4H), 1.26-1.00 (m, 2H).

δ_(C) (75.5 MHz): 139.3 (0), 129.6 (1), 128.2 (1), 126.0 (1), 69.28 (1),59.78 (2), 39.95 (2), 30.62 (2), 25.92 (2), 23.53 (2).

HRMS (electrospray mode)=192.1377940 (MH⁺) (calc. 192.1382906).

Example 8 Preparation of 2-cyclohexylpiperidin-1-ol

Activated MnO₂ (1.29 g, 14.8 mmol) was added to a solution ofN-hydroxypiperidine (0.50 g, 4.95 mmol) in dichloromethane (25 mL) at 0°C. and the mixture was stirred for 1 h. The reaction mixture wasfiltered through a pad of Celite™ and Na₂SO₄. The filtrate was addeddropwise to a solution of cyclohexylmagnesium chloride (2.0 M in diethylether, 4.95 mL, 9.90 mmol) at −10° C. The reaction mixture was stirredat 0° C. for 0.5 h and then saturated aqueous ammonium chloride solution(25 mL) and dichloromethane (25 mL) were added. The aqueous phase wasseparated and extracted with dichloromethane (2×30 mL). The combinedorganic layers were dried (Na₂SO₄) and concentrated under reducedpressure. The crude product was subjected to flash chromatography onSiO₂ eluting with 40% diethyl ether in hexanes to give the titlecompound (0.241 g, 27%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3195 (br., O—H).

δ_(H) (300 MHz): 3.32 (d, 1H, J=10.9 Hz), 2.49 (t, 1H, J=11.7 Hz), 2.16(d, 1H, J=10.4 Hz), 2.04 (t, 1H, J=12.2 Hz), 1.84-0.90 (m, 16H).

δ_(C) (75.5 MHz): 72.55 (1), 60.27 (2), 38.03 (1), 30.36 (2), 27.07 (2),26.97 (2), 26.69 (2), 25.73 (2), 23.92 (2).

HRMS (electrospray mode)=184.1690950 (MH⁺) (calc. 184.1695908).

Example 9 2,6-dimethyl-3-phenylmorpholin-4-ol

9.1. Preparation of 1-hydroxy-2,6-dimethylmorpholine

2,6-Dimethylmorpholine (26.74 mL, 0.22 mmol) was cooled to 0° C. andhydrogen peroxide (32.2 mL, 27.5%, 0.26 mmol) was added as drops withstirring. The solution was stored for six hours at 0° C. (danger ofexotherm). The product was extracted into ether and the organic phasewas separated dried (magnesium sulfate) and concentrated. Chromatographyon silica gel using ether petrol (7:3) as the eluent gave the product(17%) as mixture of syn and anti diastereoisomers, which was usedimmediately in the preparation of the nitrone.

9.2. Preparation of 2,6-dimethyl-3-phenylmorpholin-4-ol

Activated MnO₂ (0.50 g, 1.91 mmol) was added to a solution of1-hydroxy-2,6-dimethylmorpholine (0.25 g, 5.72 mmol) in dichloromethane(15 mL) at 0° C. and the mixture was stirred for 1 h. The reactionmixture was filtered through a pad of Celite™ and Na₂SO₄. The filtratewas added dropwise to a solution of phenylmagnesium chloride (2.0 M intetrahydrofuran, 1.91 mL, 3.82 mmol) at −10° C. The reaction mixture wasstirred at 0° C. for 0.5 h and then saturated aqueous ammonium chloridesolution (15 mL) and dichloromethane (15 mL) were added. The aqueousphase was separated and extracted with dichloromethane (2×20 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated underreduced pressure. The crude product was subjected to flashchromatography on SiO₂ eluting with 60% diethyl ether in hexanes to givethe title compound (0.11 g, 28%) as a white crystalline solid.

ν_(max)(Nujol™ mull)/cm⁻¹ 3325 (br., O—H).

δ_(H) (300 MHz): 7.59 (m, 2H), 7.36 (m, 3H), 4.22 (s, 1H), 4.00 (m, 2H),2.86 (m, 2H), 1.31 (d, 3H, J=6.2 Hz), 1.12 (d, 3H, J=6.6 Hz).

δ_(C) (75.5 MHz): 132.2 (1), 127.9 (1), 127.8 (1), 70.72 (1), 69.92 (1),56.57 (2), 19.05 (3), 18.36 (3).

HRMS (electrospray mode)=208.1332052 (MH⁺) (calc. 208.1329280).

Example 10 Anxiolytic Activity—Black and White Box Test

The anxiolytic effect of the compound of Example 1 (Abio 09/01) wascompared with that of the marketed anxiolytic drug, diazepam, and thecorresponding compound of formula (A), namelycis-1,2,3,4,5,6a,7,8,9,10a,10b-dodeca-1,4-oxazino-[3′,4′:2,3]benzo[d]isoxazol-4-one(hereinafter referred to as Comparative Compound A). In this test model,the effect of a stressful stimulus on the exploratory behaviour of micereceiving a control or a therapeutic amount of one of the compoundsreferred to in Table 1 was investigated.

TABLE 1 No. of Group animals Substance Dose 1 10 Vehicle 0 2 10 Diazepam1.25 mg/kg  3 10 Comparative Compound A 0.1 mg/kg 4 10 Ex. 1 (Abio09/01) 0.1 mg/kg

The black and white box comprised a Plexiglas open-topped box (45×27×27cm high) positioned on a bench 86.5 cm above the floor level in a darkand quiet room. The test box was divided into two compartments (ratio2:3) by a partition (height 60 cm). Two fifths (⅖) of the box waspainted lack and illuminated with a red light (4×15 W; 10 lux); theremainder of the box was painted white and illuminated with a 60 W (400lux) light source. The red and white lights are positioned 17 cm abovethe box. The partition is black on the side facing the black compartmentand white on the side facing the white compartment. The compartments areconnected by a 7.5 cm×7.5 cm opening in the centre bottom of thepartition. The floor of the white compartment is divided into ninefields, and the floor of the black compartment is divided into sixfields. The test room is separated into two parts by a black curtain.The drug treatment takes place in one part of the room using a minimumred light; the other part of the room, without lights, contains the testsystem.

In this test system, normal animals show a preference for exploration,measured as rearing behaviour, line crossing and time spent in the blacksection as a consequence of the aversive properties of the brightly litwhite area. The characteristic action of anxiolytic agents from thebenzodiazepine series is to disinhibit the suppressed behaviour, causinga redistribution of exploratory activity in the white section. Black andwhite box tests have been validated in mice, which are the more suitablespecies for this procedure (Costall et al., Pharm. Bioch. & Behaviour,32, 777-785 (1989)).

Naïve BKW male albino mice of 30 to 35 g were used in all studies. 10mice were normally housed in each cage and kept for at least two weekson a 12 hour light/dark cycle with lights off at 07.00 h. Behaviouraltesting was conducted between 13.00 and 18.00 h in a darkened roomilluminated with red light. Mice were taken from the dark holding roomto the testing room in an enclosed trolley and allowed at least 1 h foradaptation to the new environment.

The animals each receive a control or an active agent intraperitoneally40 minutes before testing. At the start of the test the mice are placedinto the white section of the box, facing the wall opposite to thepartition and their behaviour is recorded by remote video over a5-minute experimental period.

The results are shown in Table 2 below, wherein TB=time in whitesection; FI=Initial potency; PT=Total passages through the partition;A_(tot)=Total exploratory activity; C_(tot)=total number of crossings;R_(tot)=Total number of rearings; RB/TB=rearings in the whitecompartment/time in white; CB/TB=crossings in the white compartment/timein white; RN/TN=rearings in the black compartment/time in black;CN/TN=crossings in the black compartment/time in black.

TABLE 2 Diazepam Ex. 1 (Abio 09/01) Comparative Compound A 1.25 mg/kg0.1 mg/kg 0.1 mg/kg TB +33.2 +53.4 +52.1 FI +12.7 +11.5 +8.1 PT +1.5+0.9 +4.1 A_(tot) +46.7 +10.4 +53.7 C_(tot) +30.7 +7.5 +19.1 R_(tot)+21.2 +5 +23.9 RB/TB +0.06 +0.02 +0.04 RN/TN +0.09 +0.06 +0.13 CB/TB+0.08 −0.05 +0.01 CN/TN +0.1 0.08 +0.08

Example 11 Anxiolytic Activity—Elevated Plus Maze in Rats

The X-maze is constructed from Perspex® and comprises four arms (2 open,2 closed), each 50 cm long by 9.5 wide and 40 high (closed arms only)connected by a centre square (9.5 μg 9.5). The open arm has a small lip(3 mm) to assist in the retention of the rat on the arm. The apparatuswas housed in a room under subdued lighting conditions.

This model is based on the observation of spontaneous activity ofrodents placed in an aversive environment produced by height and oftenspaces. The open arms evoke more fears, and this results in lessexploration. Anxiolytic drugs increase the exploratory activity and thenumber of entries into the open arms. Rats were allowed at least 1 hourto acclimatize to the environment before testing commenced. Drugtreatments were given IP in a pseudo-random, blind design, 40 minutesbefore behavioural testing, as appropriate. Rats were placed onto thecentre square of the X-maze facing a specified open arm, and itsbehaviour was recorded by remote video over a 5 minute experimentalperiod. The behavioural parameters were subsequently assessed bytechnicians reviewing the videotape and included: entries into each armtype, time spent on each arm type, rearing, stretch attend postures andhead dips. The results are shown in Table 3 below.

TABLE 3 Comparative Ex. 1 Compound A (Abio 09/01) In open arms VehicleDiazepam 0.125 mg/kg 0.125 mg/kg % time spent  11 ± 4% 25 ± 5% 21 ± 11% 39 ± 10% No. of entries 1.8 ± 0.5  4 ± 0.6 3 ± 1.1 5.2 ± 1.4

Example 12 Antidepressant Activity

The “Escape deficit” test that derives from the classic paradigm of“learned helplessness” was used, as described by Meloni et al. inPharmacol. Biochem. Behav. 46, 423-6 (1993) and Gambarana et al. inBehav. Pharmacol. 6, 66-73 (1995) and Neuropsychopharmacol. 21, 247-57(1999). Exposure to an unavoidable stress induces hyperactive behaviour,which, after 24 hours, makes the rats unable to escape from anociceptive stimulus. Rats exposed to unavoidable stress make 0-5escapes out of 30 trials, while naïve rats realise 26-30 escapes.Classical anti-depressives prevent the occurrence of the escape deficitafter chronic treatment (15 days).

Rats were treated intra-peritoneally (IP) for 15 days, and thensubmitted to the unavoidable stress (16 hours after the last dosing)prior to being subjected to the escape test (24 hours after the lastdosing). The results are shown in Table 4 below.

TABLE 4 Treatment No. of escapes Naïve 24.2 ± 0.9  Vehicle 1.7 ± 0.4Comparative Compound A 3.5 ± 0.9 Ex. 1 (Abio 09/01) 23.3 ± 1.3 

Examples 10-12 Conclusions

From these results can be seen that, although the anxiolytic effects ofthe compound of this invention and the comparative prior art compound(being compounds of the same class) are similar, the success of thecompound of the invention in the test for antidepressant activity issignificantly greater than that of the comparative prior art compound.In the results reported here, the compound of the invention results inbringing the animal's behaviour back to the level of naïve animals (whohad not been subjected to the unavoidable stress).

Example 13 Antidepressant Activity—Despair and Open Field Tests

Rats treated with the compound of Example 1 were compared to either ratstreated with the classical antidepressant, clomipramine or controls inthe despair test (forced swim). The same groups were also tested for thereserpine-induced changes in open field behaviour.

13.1. Animals

Male rats of the Wistar strain (purchased from Charles River, Italy)weighing 220-240 g were used throughout all experiments. For at least 1week prior to the experiment, the rats were housed four to a cage at aconstant temperature of 21° C., and under a 12-h light/dark cycle(lights on between 08:00 and 20:00), with food and water available adlibitum.

13.2. Drugs and Treatment

A group of animals received a treatment with clomipramine hydrochloride(50 and 100 mg/kg). The drug, purchased by Sigma (USA), were freshlydiluted in physiological saline and injected intraperitoneally (IP).Rats subjected to the despair test received three such injections 24, 5and 1 hour prior to behavioural test. Physiological saline (saline) wasinjected IP to control animals. The compound of Example 1 (0.2 and 0.5mg/kg) was injected IP to a group of rats.

An acute IP injection of clomipramine hydrochloride (50 mg/kg) or of thecompound of Example 1 (0.2 and 0.5 mg/kg) or physiological saline wasmade in animals subjected to the reserpine-induced changes in the openfield test (the injection was made together with the last reserpineadministration, e.g. 1 h prior to the open field test).

All animals were gently manipulated by experienced facilities' keepersavoiding any environmental or physical stress. Rats subjected to drugsor physiological saline administration received an injection of astandard volume of solution with a 23-gauge stainless steel needle of 31mm length (for IP injections) or 21 mm length (for SC injections). Theanimals were randomly assigned to any treatment group and were used onlyonce in the behavioural experiments.

13.3. Behavioural Tests

For the despair test, rats were individually forced to swim insidevertical Plexiglas cylinders containing 15 cm of water maintained at 25°C. (Nature 266: 730-732, Porsolt et al., 1977). After 15 min in thewater they were removed and allowed to dry for 15 min in a heatedcontainer before being returned to their home cages. They were replacedin the cylinders 24 h later and the total duration of immobility wasmeasured during a 5-min test. A rat was judged to be immobile wheneverit remained passively floating in the water in a slightly hunched butupright position, its head just above the surface.

Reserpine-induced changes in open field behaviour was studied accordingto the method described in Naunyn-Schmiedeberg's Arch. Pharmacol. 293:109-114 (Vetulani et al., 1976). Reserpine (Sigma, USA) was dissolved inphysiological saline and injected daily for 14 days at the dose of 0.1mg/kg subcutaneously (SC). A period of 1 hour after the last injectionof reserpine, animals were subjected to the open field test. Thisconsisted of the measure of ambulation and rearing of the rat in acircular area divided into 17 equal sections and lighted by a centrallysuspended lamp. The areas explored by the rat at least with anteriorpaws was recorded in 5 min test. The episodes of rearing towards thecentre or the walls were also recorded in 5 min test.

13.4. Statistical Analysis

All data were analysed using univariate random design analysis ofvariance (one-way ANOVA) and the post hoc Dunnett's test for multiplecomparisons. A P-value of 0.05 or less was considered as indicative of asignificant difference. Where not indicated, one-way ANOVA revealed nosignificant level of variance.

The duration of immobility in the despair test (forced swim) in ratsinjected with physiological saline as placebo, with clomipramine 50mg/kg or with the compound of Example 1 (0.2 and 05 mg/kg) are shown inTable 5.

TABLE 5 Treatment Duration of Immobility Placebo (6) 167.60 ± 9.44 Clomipramine 50 mg/kg (4) 105.95 ± 9.13* Ex. 1 (Abio 09/01), 0.2 mg/kg(6) 167.00 ± 11.11 Ex. 1 (Abio 09/01), 0.5 mg/kg (6) 109.83 ± 9.12*Values are mean ± S.E.M. In parentheses the number of animals per eachgroup is indicated. *Significantly different as compared toplacebo-treated controls (p < 0.05, Dunnett's test for multiplecomparisons).

The number of floor units and rearing episodes in rats injected withphysiological saline as placebo, with clomipramine 50 mg/kg or with thecompound of Example 1 (0.2 and 05 mg/kg).

The number of floor units and rearing episodes in rats injected withphysiological saline as placebo, with clomipramine 50 mg/kg or with thecompound of Example 1 (0.2 and 05 mg/kg) are shown in Table 6.

TABLE 6 No. of Treatment Floor Units Rearing Episodes Reserpine +placebo (12) 131.3 ± 12.1 6.4 ± 0.2  Reserpine + clomipramine 50  84.6 ±8.1* 2.4 ± 0.2* mg/kg (7) Ex. 1 (Abio 09/01), 0.2 mg/kg (12) 128.1 ±11.9 5.9 ± 0.3  Ex. 1 (Abio 09/01), 0.5 mg/kg (12)  92.2 ± 11.1* 2.2 ±0.5* Values are mean ± S.E.M. In parentheses the number of animals pereach group is indicated. One-way ANOVA revealed no significant level ofvariance for treatment for “number of floor units” [F(3, 29) = 2.27 (p <0.10)] and for “rearing episodes” [F(3, 29) = 2.10 (p < 0.10)].*Significantly different as compared to reserpine + placebo controls (p< 0.05, Dunnett's test for multiple comparisons).

13.5. Results and Conclusions

These results show that rats treated with the compound of Example 1 at0.5 mg/kg exhibit a significant decrease of immobility time in thedespair test in comparison to the control group. This effect was similarto that of rats treated with clomipramine 50 mg/kg.

Open field performance in the reserpine test revealed a decreasedambulation (number of floor units entered) and rearing (number ofepisodes) for those rats that were treated with the compound of Example1 at 0.5 mg/kg in comparison to the control group. These effects weresimilar to those of rats treated with clomipramine 50 mg/kg.

Example 14 Anxiolytic Activity—Black and White Box Test

Further studies were conducted comparing the anxiolytic effect of thecompound of Example 1 and the compounds of Example 2-7 [Compounds P1-P6]with that of the marketed anxiolytic drug, diazepam, and thecorresponding compound of formula (A) (Comparative Compound A).

In the black/white test system (B/W box) normal animals showed apreference for remaining in the less aversive compartment, measured astime spent in black section, as well as for exploration, measured asrearing behaviour and line crossings, as a consequence of the aversiveproperties of the brightly lit. The characteristic action of anxiolyticagents is to cause a redistribution of exploratory activity and timespent in the white section.

Each mouse was placed into the centre of the white, brightly lit areabacking the opening between the B/W compartments. Animals behaviour wasrecorded by a remote video camera for 5 minutes. The recordings weresubsequently evaluated on the TV by two operators unaware of drugtreatment. The following behavioural parameters were recorded:

-   a) the latency of the initial movement from the white to the black    area: L (sec);-   b) the time spent in the white area TW (sec); the time spent in the    black area is calculated by the difference: 5−TW=TB (sec);-   c) the number of transitions between the two compartments T (No./5    min);-   d) the total activity in the white and black compartments, A_(tot)    (No./5 min);-   e) the total activity in the white area throughout the time spent in    the white area: A_(tot)W/TW (No./sec);-   f) the total activity in the black area throughout the time spent in    the black area: A_(tot)B/TB (No./sec);-   g) the total number of exploratory rearings: R_(tot) (No./5 min);-   h) the total number of line crossings: C_(tot) (No./5 min);-   i) the number of exploratory rearings in the white section RW/TW    (No./sec);-   j) the number of line crossings in the white section CW/TW    (No./sec).

Treatment was:

1 Vehicle 0

2 Diazepam 1.25 mg/kg

3 Comparative Compound A 0.1 mg/kg

4 compound of Example 1, 0.1 mg/kg

5 P1 0.1 mg/kg

6 P2 0.1 mg/kg

7 P3 0.1 mg/kg

8 P4 0.1 mg/kg

9 P5 0.1 mg/kg

10 P6 0.1 mg/kg

Each group was composed by 8 animals.

Results are shown in the following tables.

Parameter 1: Latency L (sec) Animals Mean SD SE Vehicle CMC 5.000 16.20010.2320 4.576 Diazepam 1.25 mg/kg 8.000 13.750 6.8400 2.418 P1 0.1 mg/kg8.000 20.125 15.5880 5.511 P2 0.1 mg/kg 8.000 19.625 8.4340 2.982 P5 0.1mg/kg 8.000 20.625 8.2620 2.921 P6 0.1 mg/kg 8.000 14.500 6.6550 2.353L: ANOVA comparison was not significant

Parameter 2: Time spent in White area (TW)(sec) Animals Mean SD SEVehicle CMC 5 74.200 49.7770 22.261 Diazepam 1.25 mg/kg 8 97.750 61.986021.915 P1 0.1 mg/kg 8 116.750 52.7140 18.637 P2 0.1 mg/kg 8 59.25039.0340 13.801 P5 0.1 mg/kg 8 105.125 40.6360 14.367 P6 0.1 mg/kg 8152.125 37.1580 13.137

TW: ANOVA comparison (S = significant) P-value Vehicle CMC, Diazepam1.25 mg/kg .3902 Vehicle CMC, P1 0.1 mg/kg .1245 Vehicle CMC, P2 0.1mg/kg .5844 Vehicle CMC, P5 0.1 mg/kg .2608 Vehicle CMC, P6 0.1 mg/kg.0065 S Diazepam 1.25 mg/kg, P1 0.1 mg/kg .4290 Diazepam 1.25 mg/kg, P20.1 mg/kg .1134 Diazepam 1.25 mg/kg, P5 0.1 mg/kg .7580 Diazepam 1.25mg/kg, P6 0.1 mg/kg .0277 S P1 0.1 mg/kg, P2 0.1 mg/kg .0203 S P1 0.1mg/kg, P5 0.1 mg/kg .6275 P1 0.1 mg/kg, P6 0.1 mg/kg .1447 P2 0.1 mg/kg,P5 0.1 mg/kg .0609 P2 0.1 mg/kg, P6 0.1 mg/kg .0004 S P5 0.1 mg/kg, P60.1 mg/kg .0551

Parameter 3: Transitions/5 min (T) Animals Mean SD SE Vehicle CMC 5.0009.600 6.4650 2.891 Diazepam 1.25 mg/kg 8.000 13.375 9.3490 3.306 P1 0.1mg/kg 8.000 19.500 10.8630 3.841 P2 0.1 mg/kg 8.000 10.000 9.5320 3.370P5 0.1 mg/kg 8.000 14.750 7.3050 2.583 P6 0.1 mg/kg 8.000 24.250 11.22203.968

T: ANOVA comparison P-value Vehicle CMC, Diazepam 1.25 mg/kg .4885Vehicle CMC, P1 0.1 mg/kg .0743 Vehicle CMC, P2 0.1 mg/kg .9413 VehicleCMC, P5 0.1 mg/kg .3459 Vehicle CMC, P6 0.1 mg/kg .0098 S Diazepam 1.25mg/kg, P1 0.1 mg/kg .2033 Diazepam 1.25 mg/kg, P2 0.1 mg/kg .4801Diazepam 1.25 mg/kg, P5 0.1 mg/kg .7730 Diazepam 1.25 mg/kg, P6 0.1mg/kg .0271 S P1 0.1 mg/kg, P2 0.1 mg/kg .0517 P1 0.1 mg/kg, P5 0.1mg/kg .3219 P1 0.1 mg/kg, P6 0.1 mg/kg .3219 P2 0.1 mg/kg, P5 0.1 mg/kg.3219 P2 0.1 mg/kg, P6 0.1 mg/kg .0046 S P5 0.1 mg/kg, P6 0.1 mg/kg.0517

Parameter 4: Total Activity: A_(tot) (No./5 min) Animals Mean SD SEVehicle CMC 5.000 112.400 49.1710 21.990 Diazepam 1.25 mg/kg 8.000213.125 90.5810 32.025 P1 0.1 mg/kg 8.000 197.375 47.7670 16.888 P2 0.1mg/kg 8.000 168.625 53.6020 18.951 P5 0.1 mg/kg 8.000 197.000 37.849013.382 P6 0.1 mg/kg 8.000 229.375 42.1660 14.908

A_(tot): ANOVA comparison P-value Vehicle CMC, Diazepam 1.25 mg/kg .0035S Vehicle CMC, P1 0.1 mg/kg .0123 S Vehicle CMC, P2 0.1 mg/kg .0902Vehicle CMC, P5 0.1 mg/kg .0126 S Vehicle CMC, P6 0.1 mg/kg .0008 SDiazepam 1.25 mg/kg, P1 0.1 mg/kg .5821 Diazepam 1.25 mg/kg, P2 0.1mg/kg .1250 Diazepam 1.25 mg/kg, P5 0.1 mg/kg .5732 Diazepam 1.25 mg/kg,P6 0.1 mg/kg .5702 P1 0.1 mg/kg, P2 0.1 mg/kg .3173 P1 0.1 mg/kg, P5 0.1mg/kg .9895 P1 0.1 mg/kg, P6 0.1 mg/kg .2664 P2 0.1 mg/kg, P5 0.1 mg/kg.3236 P2 0.1 mg/kg, P6 0.1 mg/kg .0386 S P5 0.1 mg/kg, P6 0.1 mg/kg.2610

Parameter 5: Total Activity in white area: A_(tot)W/TW (No./sec) AnimalsMean SD SE Vehicle CMC 5.000 .475 .1800 .080 Diazepam 1.25 mg/kg 8.000.723 .3030 .107 P1 0.1 mg/kg 8.000 .608 .2980 .105 P2 0.1 mg/kg 8.000.519 .2530 .089 P5 0.1 mg/kg 8.000 .571 .1570 .055 P6 0.1 mg/kg 8.000.705 .1380 .049 A_(tot)W/TW: ANOVA comparison was not significant

Parameter 6: Total Activity in black area: A_(tot)B/TB (No./sec) AnimalsMean SD SE Vehicle CMC 5.000 .341 .1450 .065 Diazepam 1.25 mg/kg 8.000.723 .3190 .113 P1 0.1 mg/kg 8.000 .652 .1380 .049 P2 0.1 mg/kg 8.000.571 .1840 .065 P5 0.1 mg/kg 8.000 .708 .1530 .054 P6 0.1 mg/kg 8.000.835 .1750 .062

A_(tot)B/TB: ANOVA comparison P-value Vehicle CMC, Diazepam 1.25 mg/kg.0017 S Vehicle CMC, P1 0.1 mg/kg .0091 S Vehicle CMC, P2 0.1 mg/kg.0496 S Vehicle CMC, P5 0.1 mg/kg .0025 S Vehicle CMC, P6 0.1 mg/kg<.0001 S Diazepam 1.25 mg/kg, P1 0.1 mg/kg .4832 Diazepam 1.25 mg/kg, P20.1 mg/kg .1351 Diazepam 1.25 mg/kg, P5 0.1 mg/kg .8796 Diazepam 1.25mg/kg, P6 0.1 mg/kg .2663 P1 0.1 mg/kg, P2 0.1 mg/kg .4183 P1 0.1 mg/kg,P5 0.1 mg/kg .5817 P1 0.1 mg/kg, P6 0.1 mg/kg .0740 P2 0.1 mg/kg, P5 0.1mg/kg .1774 P2 0.1 mg/kg, P6 0.1 mg/kg .0115 S P5 0.1 mg/kg, P6 0.1mg/kg .2080

Parameter 7: R_(tot) (No./5 min) Animals Mean SD SE Vehicle CMC 5.00022.600 19.6800 8.801 Diazepam 1.25 mg/kg 8.000 68.750 31.4950 11.135 P10.1 mg/kg 8.000 60.375 23.8860 8.445 P2 0.1 mg/kg 8.000 60.250 28.709010.150 P5 0.1 mg/kg 8.000 69.500 20.3960 7.211 P6 0.1 mg/kg 8.000 75.62511.6120 4.105

R_(tot): ANOVA comparison P-value Vehicle CMC, Diazepam 1.25 mg/kg .0016S Vehicle CMC, P1 0.1 mg/kg .0083 S Vehicle CMC, P2 0.1 mg/kg .0085 SVehicle CMC, P5 0.1 mg/kg .0013 S Vehicle CMC, P6 0.1 mg/kg .0004 SDiazepam 1.25 mg/kg, P1 0.1 mg/kg .4860 Diazepam 1.25 mg/kg, P2 0.1mg/kg .4795 Diazepam 1.25 mg/kg, P5 0.1 mg/kg .9501 Diazepam 1.25 mg/kg,P6 0.1 mg/kg .5669 P1 0.1 mg/kg, P2 0.1 mg/kg .9917 P1 0.1 mg/kg, P5 0.1mg/kg .4480 P1 0.1 mg/kg, P6 0.1 mg/kg .2078 P2 0.1 mg/kg, P5 0.1 mg/kg.4419 P2 0.1 mg/kg, P6 0.1 mg/kg .2042 P5 0.1 mg/kg, P6 0.1 mg/kg .6098

Parameter 8: C_(tot) (No./5 min) Animals Mean SD SE Vehicle CMC 5.00089.800 30.0870 13.455 Diazepam 1.25 mg/kg 8.000 144.375 60.4650 21.378P1 0.1 mg/kg 8.000 137.000 25.7570 9.107 P2 0.1 mg/kg 8.000 108.37527.7950 9.827 P5 0.1 mg/kg 8.000 127.500 20.3890 7.209 P6 0.1 mg/kg8.000 153.750 32.9490 11.649

C_(tot): ANOVA comparison P-value Vehicle CMC, Diazepam 1.25 mg/kg .0107S Vehicle CMC, P1 0.1 mg/kg .0258 S Vehicle CMC, P2 0.1 mg/kg .3673Vehicle CMC, P5 0.1 mg/kg .0717 Vehicle CMC, P6 0.1 mg/kg .0032 SDiazepam 1.25 mg/kg, P1 0.1 mg/kg .6820 Diazepam 1.25 mg/kg, P2 0.1mg/kg .0508 Diazepam 1.25 mg/kg, P5 0.1 mg/kg .3506 Diazepam 1.25 mg/kg,P6 0.1 mg/kg .6027 P1 0.1 mg/kg, P2 0.1 mg/kg .1171 P1 0.1 mg/kg, P5 0.1mg/kg .5979 P1 0.1 mg/kg, P6 0.1 mg/kg .3542 P2 0.1 mg/kg, P5 0.1 mg/kg.2909 P2 0.1 mg/kg, P6 0.1 mg/kg .0152 S P5 0.1 mg/kg, P6 0.1 mg/kg.1497

Parameter 9: RW/TW (No./sec) Animals Mean SD SE Vehicle CMC 5.000 .083.0810 .036 Diazepam 1.25 mg/kg 8.000 .124 .0930 .033 P1 0.1 mg/kg 8.000.155 .1060 .038 P2 0.1 mg/kg 8.000 .099 .0900 .032 P5 0.1 mg/kg 8.000.123 .0870 .031 P6 0.1 mg/kg 8.000 .190 .0500 .018 RW/TW: ANOVAcomparison was not significant

Parameter 10: CW/TW (No./sec) Animals Mean SD SE Vehicle CMC 5.000 .391.1150 .052 Diazepam 1.25 mg/kg 8.000 .599 .2330 .082 P1 0.1 mg/kg 8.000.454 .1990 .071 P2 0.1 mg/kg 8.000 .420 .1790 .063 P5 0.1 mg/kg 8.000.448 .0840 .030 P6 0.1 mg/kg 8.000 .515 .1040 .037 CW/TW: ANOVAcomparison was not significant

1. A method of treating anxiety or depression comprising administeringto a subject having anxiety or depression a therapeutically effectiveamount of a compound of formula (I):

in which— X represents O or CH₂; R³ and R⁴ each independently representhydrogen or C₁₋₆ alkyl; p represent 0 or 1; and R represents a five- orsix-membered saturated or unsaturated ring selected from:

or R represents a five- or six-membered substituted or unsubstitutedunsaturated ring selected from:

wherein R¹ and R² together represent an oxo group, or R¹ and R² eachrepresent hydrogen, methoxy or ethoxy, or R¹ and R² together with theinterjacent carbon atom represent a 1,3-dioxolane or 1,3-dioxane ring,attached via the 2 position and optionally bearing one or more methyl orethyl groups; or a tautomer thereof; or a salt thereof.
 2. A method asclaimed in claim 1 in which R represents a five- or six-memberedsubstituted or unsubstituted unsaturated ring selected from:

wherein R¹ and R² together represent an oxo group, or R¹ and R² eachrepresent methoxy or ethoxy, or R¹ and R² together with the interjacentcarbon atom represent a 1,3-dioxolane or 1,3-dioxane ring, attached viathe 2 position and optionally bearing one or more methyl or ethylgroups, and p represents
 0. 3. A method as claimed in claim 1 in whichR¹ and R² together represent an oxo group.
 4. A method as claimed inclaim 1 in which R represents a five- or six-membered substituted orunsubstituted unsaturated ring selected from:

and X is O.
 5. A method as claimed in claim 1 in which R³ and R⁴ areidentical.
 6. A method as claimed in claim 1 in which R³ and R⁴ eachrepresents hydrogen.
 7. A method as claimed in claim 1 wherein thecompound is of formula (II):

in which— R³ and R⁴ each independently represent hydrogen or C₁₋₆ alkyl;p represent 0 or 1; and R represents a five- or six-membered saturatedor unsaturated ring selected from:

or R represents a five- or six-membered substituted or unsubstitutedunsaturated ring selected from:

wherein R¹ and R² together represent an oxo group, or R¹ and R² eachrepresent hydrogen, methoxy or ethoxy, or R¹ and R² together with theinterjacent carbon atom represent a 1,3-dioxolane or 1,3-dioxane ring,attached via the 2 position and optionally bearing one or more methyl orethyl groups; or a tautomer thereof; or a salt thereof.
 8. A method asclaimed in claim 7 wherein the compound is of formula (III):

in which— R¹ and R² each represent hydrogen or together represent an oxogroup; or a salt thereof.
 9. A method as claimed in claim 1 wherein thecompound is of formula


10. A method as claimed in claim 1 wherein the subject has anxiogenesiscaused by withdrawal from benzodiazepines, or caused by abruptly ceasingthe administration of drugs of abuse.